氧合硫胺素(OT),抗代谢物的类似物,可以通过在体外和体内引起G1期停滞来抑制核糖的非氧化合成并诱导细胞凋亡。然而,分子机制尚不清楚。在本研究中,使用改良的SILAC方法(mSILAC)进行了定量蛋白质组学分析,以确定代谢抑制对用不同剂量的OT处理的MIAPaCa-2癌细胞中蛋白质表达动态变化的影响(0μM,5μM,50μM和500μM)和时间点(0h,12h和48h)。在用OT处理的MIAPaCa-2细胞中总共鉴定了52种差异蛋白,包括14种磷酸化蛋白。基于动态表达模式,这些蛋白质分为三个簇,直接下调(簇1,占总蛋白质的37%),直立“V”形表达模式(第2组,总计47.8%),和右“V”形图案(第3组,总计15.2%)。其中,OT处理以时间依赖性方式显著下调膜联蛋白A1表达,而在OT剂量依赖性方式中没有观察到该蛋白的变化。通路分析表明,转酮醇酶的抑制导致与细胞凋亡相关的多个细胞信号通路的变化。蛋白质的时间表达模式显示,OT通过抑制磷激酶表达以时间依赖性方式改变蛋白质表达的动力学,导致癌细胞凋亡。这项研究的结果表明,单个代谢酶活性的干扰改变了多个细胞信号传导途径。
Oxythiamine (OT), an analogue of anti-metabolite, can suppress the nonoxidative synthesis of ribose and induce cell apoptosis by causing a G1 phase arrest in vitro and in vivo. However, the molecular mechanism remains unclear yet. In the present study, a quantitative proteomic analysis using the modified SILAC method (mSILAC) was performed to determine the effect of metabolic inhibition on dynamic changes of protein expression in MIA PaCa-2 cancer cells treated with OT at various doses (0 μM, 5 μM, 50 μM and 500 μM) and time points (0 h, 12 h and 48 h). A total of 52 differential proteins in MIA PaCa-2 cells treated with OT were identified, including 14 phosphorylated proteins. Based on the dynamic expression pattern, these proteins were categorized in three clusters, straight down-regulation (cluster 1, 37% of total proteins), upright \"V\" shape expression pattern (cluster 2, 47.8% total), and downright \"V\" shape pattern (cluster 3, 15.2% total). Among them, Annexin A1 expression was significantly down-regulated by OT treatment in time-dependent manner, while no change of this protein was observed in OT dose-dependent fashion. Pathway analysis suggested that inhibition of transketolase resulted in changes of multiple cellular signaling pathways associated with cell apoptosis. The temporal expression patterns of proteins revealed that OT altered dynamics of protein expression in time-dependent fashion by suppressing phosphor kinase expression, resulting in cancer cell apoptosis. Results from this study suggest that interference of single metabolic enzyme activity altered multiple cellular signaling pathways.